Monoclonal antibodies are of great laboratory and therapeutic use. Antibody derivatives with engineered site-specific fluorescence or binding properties have been developed and used for many years. More recently, antibodies have been also developed as therapeutic agents, currently presenting the fastest growing class of pharmaceuticals [1]. Antibodies are multidomain proteins of two light and two heavy chains held together by disulfide bonds. The variable regions specify binding to a particular antigen, and part of the constant regions is responsible for effector functions via binding to Fc receptors on the surface of immune cells. Because of their potential in the cure of various diseases, antibodies currently constitute the most rapidly growing class of human therapeutics (Carter. Nature Reviews Immunology. 2006, 6(5), 343). Since 2001, their market has been growing at an average yearly growth rate of 35%, the highest rate among all categories of biotech drugs (S. Aggarwal. Nature. BioTech. 2007, 25 (10) 1097).
Engineering of antibody conjugates has further increased the versatility of antibody applications. In many laboratory techniques, enzymes or fluorescent probes are conjugated to antibodies to carry out an assay function, for example quantitation of antigen abundance. In cases of targeted therapy, toxic small molecules are attached to antibodies that specifically bind biomarkers on diseased cells [2-4]. Various approaches to antibody conjugation have been pursued, for example attachment to surface lysines [5], to Fc carbohydrates [6], or to partially reduced interchain disulfides [7].
Antibody conjugation to engineered surface cysteine remains a very attractive option because most antibodies do not have cysteines other than the ones consumed in intra- and inter-chain disulfide bonds. Small molecules can be attached at the specific site of cysteine substitution via a thiol reactive chemistry such as maleimides [8-14]. Engineering in the CH1 and CH3 domains has been favored to avoid interference with antigen binding of the variable regions and effector function of CH2. Different criteria for successful antibody conjugation via engineered cysteines have been considered. For example, the antibody domain in which to carry out mutation, the exposure of the mutated site, the amino acid to be substituted are several of the variables to take into account. A high throughput screening approach to identifying sites suitable for cysteine engineering and conjugation has been developed [15]. Two of the most common problems associated with antibody cysteine variants are oligomerization and poor labeling. Yet, there is no universal tool for predicting whether an antibody cysteine variant will be stable and efficiently conjugated. Furthermore, cysteine variants currently exist only for the CL, CH1 and CH3 domains [8, 9, 11, 12, 15].
Thus, there is a need for additional immunoglobulin cysteine variants that can be used in the generation of stable immunoglobulin conjugates.